The Effects of Bisphenol A on Human Male Infertility: A Review of Current Epidemiological Studies

Endocrine disruptor chemicals (EDCs) can have a harmful effect on the human body’s endocrine system and thus adversely affect the development, reproduction, neurological, cardiovascular, and immune systems and metabolism in humans and wildlife. According to the World Health Organization, EDCs are mostly man-made and found ubiquitously in our daily lives, notably in pesticides, metals, and additives or contaminants in food and personal care products. Human exposure occurs through ingestion, inhalation, and dermal contact. Bisphenol A (BPA) is a proven EDC capable of mimicking or blocking receptors and altering hormone concentrations and metabolism. Although consumed in low doses, it can stimulate cellular responses and affect the body’s functions. In humans, exposure to BPA has been correlated with the onset or development of several diseases. This literature review aimed to verify the effects of BPA on human male infertility using the most recently published literature. Thus, this review allowed us to conclude that this compound seems to have harmful effects on human male fertility, causing changes in hormonal and semen characteristics. However, these conclusions lack more robust and reproducible scientific studies. Even so, and since male infertility prevalence is increasing, preventive measures must be taken to ensure male fertility.


Introduction
Endocrine disruptor chemicals (EDC) can have a harmful effect on the endocrine system [1]. They are defined by the Environmental Protection Agency of the United States of America as "an agent that interferes with the synthesis, secretion, transportation, binding or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development and/or behaviour" [2,3]. By interfering with the hormonal system, they adversely affect the development, reproduction, neurological, cardiovascular, immune, and metabolism systems in both humans and wildlife. These effects can be observed after long exposure and may also have consequences for the next generation, i.e., they may vary depending on the time of exposure and the hormonal balance of the exposed individual, which is determined by age, sex, and other factors [1].
According to the World Health Organization (WHO), EDs are mostly man-made and are found in various materials such as pesticides, metals, and additives or contaminants in food and personal care products. Human exposure occurs through ingesting food, dust, and water and inhaling gases and particles in the air and through the skin. There may also be passage to the foetus through the placenta and to children through breast milk [1]. More specifically, adults come into contact mainly through ingestion of contaminated drinking water, meat, and fatty dairy products and inhalation of polluted air, while babies are exposed through breastfeeding, contact with baby products, and also by inhalation of polluted air [2].
EDs are highly heterogeneous and can be classified as natural and synthetic or grouped according to their origin [2,4]. They are also considered "chemical chameleons", i.e., they At room temperature, BPA is a solid, white, crystalline substance w that has a molecular weight of 228.29 g/cm 3 , a melting point of 156 °C, a of 220 °C (at a pressure of 5 hPa). In addition, it has an octanol-water pa of log Pow = 3.32, indicating that it has a good solubility in fats and, the ubility in water (~200 mg/dm 3 at 25 °C). The presence of hydroxyl groups good reactivity. Like other phenols, BPA can be converted into ethers [11].
BPA does not occur freely in nature, and when exposed to air, it u oxidation and degradation, resulting in a low half-life (0.2 days) [6]. At room temperature, BPA is a solid, white, crystalline substance with a mild odour that has a molecular weight of 228.29 g/cm 3 , a melting point of 156 • C, and a boiling point of 220 • C (at a pressure of 5 hPa). In addition, it has an octanol-water partition coefficient of log Pow = 3.32, indicating that it has a good solubility in fats and, therefore, a low solubility in water (~200 mg/dm 3 at 25 • C). The presence of hydroxyl groups determines their good reactivity. Like other phenols, BPA can be converted into ethers, esters, and salts [11].
BPA does not occur freely in nature, and when exposed to air, it undergoes photooxidation and degradation, resulting in a low half-life (0.2 days) [6].

Production and Application
The presence of BPA in the environment is linked only to anthropogenic activity, being considered one of the most produced chemicals worldwide [8,11]. Global BPA production gradually increased from 5 to 8 million tons between 2010 and 2016 and is estimated to reach 10.2 million by 2023. There has been a prominent increase in production in the emerging markets of China, India, Russia, and Mexico [6]. Due to its mass production, many derivatives of this substance are released into the environment, thus contributing to an increase in pollution and contamination of soils and groundwater [13].
In the 1950s, it was observed that BPA could be polymerised to make polycarbonate plastic, a miraculous product that is cheap, lightweight, transparent, and colourable; resistant to impact, heat, and chemicals; unalterable over time; and easy to mould and thermoform [14]. The manufacturing process involves a reaction between acetone and two phenols in a continuous, closed process under certain temperature conditions using an acid catalyst. The substitution of atoms in different numbers and positions results in the formation of analogous compounds with specific characteristics intended for various industrial applications [15].
It is currently used as a material to produce phenol resins, polyacrylics, and polyesters, but it is mainly to produce epoxy resins and polycarbonate plastics. Epoxy resins are used as coatings on canning cans and metal bottle covers, as protective coatings and finishes, in automobile parts, adhesives, and aerospace applications, and as a coating for PVC pipes. Polycarbonate plastics are used to manufacture automotive lenses, household appliances, food packaging, plastic bottles, pacifiers, and even toys [10]. BPA has also been used since the 1960s as a component of many dental restoration materials [16]. Recently, its uses have expanded to producing optical and electronic materials [17].
Despite all the applications, consumers' concern about the possible consequences for humans has resulted in "BPA free" products and the development of bisphenol analogues to replace BPA. These compounds (e.g., bisphenol S, bisphenol F, bisphenol AF, bisphenol E, and many others) have been extensively used in numerous everyday products, and humans are persistently exposed to these compounds after they are released from the products. Unfortunately, these other derivatives also seem to have similar harmful effects, and some of them are even more toxic. Specifically, bisphenol S and bisphenol F were found to have similar hormonal activity as BPA (estrogenic, antiestrogenic, androgenic, and antiandrogenic activities) and also alter organ weights, reproductive endpoints, and enzyme expression both in vitro and in vivo [18][19][20]. Thus, BPA analogues have also been considered as EDCs and may pose a human health risk similar to BPA.

Sources of Exposure
The degree of exposure to BPA can vary dramatically depending on various socioeconomic factors, lifestyle, comorbidities, and routes of exposure, like food, water, air, and soil ( Figure 2) [11].

Food
The main source of BPA contamination in humans is through food [9,10,21]. Its pres ence in edible products is related to the exposure of animals and raw materials to BPA the accumulation of BPA in the environment, and the contact of food with polymers con taining this substance [11]. Direct contamination of food is caused by the migration of BPA present in the containers in which food is stored. Epoxy resins produced with BPA are

Food
The main source of BPA contamination in humans is through food [9,10,21]. Its presence in edible products is related to the exposure of animals and raw materials to BPA, the accumulation of BPA in the environment, and the contact of food with polymers containing this substance [11]. Direct contamination of food is caused by the migration of BPA present in the containers in which food is stored. Epoxy resins produced with BPA are used as a coating on the surfaces of metal cans that are in contact with food and beverages, while polycarbonate plastics are used in containers that transport/store food and beverages [10]. The European Food Safety Authority (EFSA) has recently updated the tolerable daily intake of BPA from 4 µg to 0.2 ng per kilogram of body weight per day [22,23]. The previous TDI was temporary due to uncertainties in the evidence, whereas the revised EFSA decision was made according to new scientific evidence that established BPA as a health risk concern since, in all age groups, average and high exposure to BPA exceeded the 0.2 ng per kilogram of body weight per day [23]. On the other hand, the Environmental Protection Agency of the United States of America sets the tolerable value daily at 50 µg/kg/day [24]. Moreover, according to the European Commission, the migration of BPA into food from varnishes or coatings applied to materials and objects should not exceed a specific migration limit of 0.05 mg of BPA per kilogram of food. However, the migration of BPA is not permitted from varnishes or coatings applied to materials and articles specifically intended to be in contact with infant formulae, processed cereal-based foods, baby foods, special medical foods developed to meet the nutritional requirements of infants and young children, and milk beverages and similar products specifically intended for young children, as referred to in Regulation (EU) No 609/2013 [22,23]. BPA can migrate from plastics after washing and sterilisation in alkaline solutions or in hot water (e.g., steam) [10]. The migration ratio of BPA from polycarbonate bottles to other solutions varies according to their chemical properties. Temperature and, particularly, the prolonged use of bottles increases the hydrolysis of the polymer, causing a more intense migration to water [11]. BPA is authorised to be used as a monomer in plastic food contact materials in accordance with Commission Regulation (EU) No 10/2011. In addition, Commission Implementing Regulation (EU) No 321/2011, which amends the abovementioned agreement, imposes a restriction on the use of BPA in the manufacture of baby bottles for children, in accordance with the precautionary principle [22,23].

Water
BPA can be found in the aquatic environment and has been detected at high levels in leachates from landfills or factories, especially those that manufacture and process plastics [10]. In surface waters, it is usually found in low concentrations; however, near waste deposits contaminated with this substance or waste deposits of plastic materials, the concentrations are much higher [11]. BPA can also be detected in river waters; it can be degraded in aerobic conditions but not in anaerobic conditions. Despite the degradation of BPA in freshwater by bacteria and sunlight, its half-life of 3 to 5 days may be sufficient to cause harmful effects on aquatic organisms due to the metabolites that persist. Interestingly, BPA can persist longer in seawater than in river water without any degradation (about 30 days), and the bio-available fraction of dissolved BPA can increase with salinity. Thus, the possibility of contamination of a marine organism may be greater than a freshwater organism. Therefore, from the aquatic environment, the ingestion of freshwater fish or seafood contaminated with BPA may be the main route of contamination for humans [10,25]. Bacteria and other microorganisms that effectively degrade this substance may be useful in its removal from the polluted environment [11].

Air
Inhalation is the second main source of exposure [9]. BPA is emitted into the atmosphere during industrial activity, where it can remain due to its adhesion to particulate matter. The possibility of inhaling high levels of BPA through the air is very low [6, 10,11]. However, due to the large quantities produced annually, workers in companies engaged in the production of plastics are an exception [16]. Various household items manufactured from BPA, such as epoxy-based flooring and electronic equipment, can release and volatilise this compound with prolonged use. Consequently, BPA can accumulate in household dust and be inhaled [9].

Soil
The main source of BPA in soils is the terrestrial application of sewage or bio-solid sludge and human waste. BPA released into groundwater or surface water can be absorbed into soil or sediment. It was found that there are higher levels of BPA in sediments than in surface waters. However, soil contamination can be correlated with population density due to the increase in human waste contaminated with BPA, such as household and/or industrial waste [10,25].
BPA is thought to have a moderate affinity with soil organic matter and thus is unlikely to be mobile or bio-available in soils. However, mobility can be affected by soil chemistry and texture. There is evidence of an increase in BPA sorption in the presence of iron, cadmium, and lead and rapid and complete desorption in sandy and acidic soils [25].

Conjugation, Metabolism, and Excretion
In humans, orally ingested BPA is rapidly and efficiently absorbed through the gastrointestinal tract (>95% of the dose) and subsequently metabolised in the liver, which plays an essential role in the metabolism of this substance ( Figure 2) [8,9,26]. Its glucuronidation, the main biotransformation pathway, is a detoxification reaction that is carried out by uridine diphosphate glycuronosyltransferase (UGT), a glycosyltransferase that is present in the liver. UGT in the human foetal liver is present at a concentration five times lower than in the adult liver; this means that a foetus may face a higher risk of deleterious effects [9,10]. This process increases the solubility of BPA in water, which leads to faster excretion in the urine (half-life of 5.4-6.4 h) [27].
BPA glucuronide, with little or no estrogenic activity, is the main metabolite found in urine, while free or unconjugated BPA, considered the active form, is the main component present in faeces [10,26]. More than 99% of free BPA and its metabolites are excreted in faeces and urine, and less than 1% accumulates in tissues [10]. Some studies suggest that there may be gender differences in metabolite concentrations in the urine, with women having higher levels of BPA sulphate and men having higher levels of BPA glucuronide [8,10].
BPA is also conjugated in vivo in BPA sulphate by sulfotransferases found in the liver. BPA sulfation eliminates its estrogenic activity [8,26].
However, unlike dietary exposure, almost all BPA resulting from transdermal exposure prevents hepatic metabolism, resulting in significantly higher concentrations of the unconjugated form (free BPA) in the bloodstream [27].

Detection in Biological Samples
Given the prevalence of BPA in our environment, it is not surprising that measurable levels have been detected in most individuals who were analysed. It is possible to detect the presence of BPA in human tissues and fluids, including maternal blood (0.3-18.9 ng/mL), maternal urine (31.9 µg/L), amniotic fluid (median = 0.26 ng/mL), placental tissue (median = 12.7 ng/g), umbilical cord blood (0.2-9.2 ng/mL), breast milk (0.61-0.7 µg/L), and human colostrum (3.41 ng/mL) [7,11,16]. Concentrations of unconjugated BPA in human serum have been detected at levels ranging from 0.2 to 20 ng/mL [16]. Depending on the pathway by which the individual is exposed, BPA levels in the circulation may also be different [8]. As most of the BPA consumed orally is excreted in the urine (concentration varies between 1.6 and 946 µg/L) in less than 24 h after consumption, and the expected concentrations in the blood are very low, urine is the preferred body fluid for estimating BPA exposure in humans [26].
Several studies have examined BPA levels in the serum of pregnant women, umbilical cord blood, and foetal plasma. The results of these studies indicate that this substance crosses the placental barrier [28]. Of particular concern are the high levels detected mainly in developmental stages with a possible higher sensitivity to BPA [16]. BPA can accumulate in foetuses due to lower metabolic clearance. After swallowing amniotic fluid, BPA can be conjugated by the foetal liver. Thus, a foetus is susceptible to BPA exposure throughout development and may be exposed to even higher levels than those measured in the adult's blood. Another important consideration for the health of the newborn is the potential exposure to BPA in breast milk since it is a compound with lipophilic properties [16]. By being lipophilic, it can accumulate in adipose tissue, including fat mammary glands, and cause dysregulation in various metabolic processes, both in the baby and in the adult [9,11].

Mechanisms of Action
BPA, with a structure similar to 17β-oestradiol (E2), binds to both the alpha (ERα) and the beta oestrogen receptors (ERβ), with approximately 10 times more affinity for ERβ. This EDC can also bind to the G-protein-coupled oestrogen receptor (GPER), the gamma peroxisome proliferator-activated receptor (PPAR-γ), and the orphan nuclear oestrogen gamma receptor (ERR-γ) [29]. Binding to these receptors can lead to other changes in cells and tissues, not just endocrine disruptions [27].
It is now widely accepted that BPA not only has the effectiveness of E2 but is also potent in relation to several of its effects, that is, it has a potent estrogenic effect [8]. In addition, it can act as an anti-oestrogen, blocking the oestrogen response by competing with endogenous E2. It can also bind directly to androgen receptors (AR), possibly being anti-androgenic, blocking endogenous androgen action [30].
Endogenous oestrogens have various effects at the biological level, from every organism to the systems of organs, cells, and gene expression [8]. They play a key role in the testicle since their biosynthesis occurs in the testicular cells. The absence of ERs causes adverse effects on spermatogenesis and steroid synthesis. Physiological levels of E2 are essential for normal spermatogenesis, and when altered, it can lead to the occurrence of pathologies. For example, in infertility, there is an excess of E2 together with a decrease in testosterone [31]. In addition to its estrogenic activity, there is some evidence that BPA binds to the thyroid hormone receptor, acting as an antagonist and preventing the binding of T3 [8]. The affinity for the thyroid receptor is lower than the affinity for the oestrogen receptor, suggesting that elevated BPA levels would be needed to antagonise the hormonal action of the thyroid [7].
Regarding the male reproductive system, this substance perturbates the hypothalamicpituitary-testicle axis and modulates gene expression and enzymatic activity of testicular steroidogenesis ( Figure 3). It is also associated with a decrease in the activity of the antioxidant system, resulting in an increase in oxidative stress, the most common cause of sperm damage [19]. It exerts multidirectional effects as it interacts with several receptors, generates reactive oxygen species, alters cell signalling, causes mutagenic changes, and inhibits DNA methylation [11].
receptor, suggesting that elevated BPA levels would be needed to antagonise the hormonal action of the thyroid [7]. Regarding the male reproductive system, this substance perturbates the hypotha lamic-pituitary-testicle axis and modulates gene expression and enzymatic activity of tes ticular steroidogenesis ( Figure 3). It is also associated with a decrease in the activity of the antioxidant system, resulting in an increase in oxidative stress, the most common cause o sperm damage [19]. It exerts multidirectional effects as it interacts with several receptors generates reactive oxygen species, alters cell signalling, causes mutagenic changes, and inhibits DNA methylation [11].
Although the mechanisms of action are not yet fully understood, BPA also interferes with other functions of the body, including the development of the central nervous sys tem, the endocrine action of the pancreas, the immune system, and the function of sex hormones, insulin, leptin, and adiponectin. These BPA effects may also be responsible for the development of several types of cancer, such as breast, prostate, pancreas, pituitary cerebellar cortex, and heart cancer [11,30].

Methods
For the preparation of this review, a bibliographic search was conducted from Sep tember 2022 to January 2023, using the search databases PubMed, Scopus, and Google Scholar. The following items were used as keywords: (bisphenol A) AND (endocrine dis ruptor); (bisphenol A) AND (reproduction); (bisphenol A) AND (male reproductive sys Although the mechanisms of action are not yet fully understood, BPA also interferes with other functions of the body, including the development of the central nervous system, the endocrine action of the pancreas, the immune system, and the function of sex hormones, insulin, leptin, and adiponectin. These BPA effects may also be responsible for the development of several types of cancer, such as breast, prostate, pancreas, pituitary, cerebellar cortex, and heart cancer [11,30].

Methods
For the preparation of this review, a bibliographic search was conducted from September 2022 to January 2023, using the search databases PubMed, Scopus, and Google Scholar. The following items were used as keywords: (bisphenol A) AND (endocrine disruptor); (bisphenol A) AND (reproduction); (bisphenol A) AND (male reproductive system); (bisphenol A) AND (male reproduction OR male fertility OR male infertility); (bisphenol A) AND (spermatogenesis OR sperm). Priority was given to articles from the last ten years to study the consequences of BPA on male infertility; however, older articles that were considered relevant were not excluded. The inclusion criteria comprised epidemiological studies regarding male infertility that related BPA to hormonal changes and semen characteristics. The exclusion criteria included all articles that were duplicates, unrelated, inaccessible, and not written in English. The search strategy is described in Figure 4.

Effects on Human Male Fertility
Widespread and ongoing exposure to BPA in humans was confirmed using biomonitoring studies in the general population, in which more than 80% of participants had detectable levels in the urine [32]. Analysis of data from the available literature indicates that the reproductive system is one of the targets of BPA toxicity. This substance acts mainly by mimicking the effect of oestrogen, modifying DNA methylation, and modulating enzymatic activities, resulting in metabolic diseases, spermatogenesis defects and/or infertility in men. The deleterious effect on male reproductive function can occur during embryonic, pubertal, and/or adult life [33].
The disrupting endocrine effects of BPA on male reproductive health have raised concerns over the past few decades. Studies report that exposure to this compound is associated with reduced sperm count, worsening of sperm motility and speed, decreased epididymis weight, poor insulin signalling and glucose homeostasis, and decreased testicle steroids, follicle-stimulating hormone (FSH), and testosterone (T) levels in the serum. However, these observations are based on animal models and in vitro studies [34][35][36][37][38].
Regarding the effects of BPA on the human male reproductive system, studies are still limited, and the underlying molecular mechanisms remain uncertain.
Infertility is one of the main health problems that affect human sociocultural life and is defined, according to the WHO, as "the inability to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse" [39]. There are two types: pri-

Effects on Human Male Fertility
Widespread and ongoing exposure to BPA in humans was confirmed using biomonitoring studies in the general population, in which more than 80% of participants had detectable levels in the urine [32]. Analysis of data from the available literature indicates that the reproductive system is one of the targets of BPA toxicity. This substance acts mainly by mimicking the effect of oestrogen, modifying DNA methylation, and modulating enzymatic activities, resulting in metabolic diseases, spermatogenesis defects and/or infertility in men. The deleterious effect on male reproductive function can occur during embryonic, pubertal, and/or adult life [33].
The disrupting endocrine effects of BPA on male reproductive health have raised concerns over the past few decades. Studies report that exposure to this compound is associated with reduced sperm count, worsening of sperm motility and speed, decreased epididymis weight, poor insulin signalling and glucose homeostasis, and decreased testicle steroids, follicle-stimulating hormone (FSH), and testosterone (T) levels in the serum. However, these observations are based on animal models and in vitro studies [34][35][36][37][38].
Regarding the effects of BPA on the human male reproductive system, studies are still limited, and the underlying molecular mechanisms remain uncertain.
Infertility is one of the main health problems that affect human sociocultural life and is defined, according to the WHO, as "the inability to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse" [39]. There are two types: primary infertility, which refers to couples who have never had a child, and secondary, which refers to infertile couples who have already managed to become pregnant at least once [40]. It affects 8 to 15% of couples worldwide, with an expected tendency to increase in the coming years, and about 50% of cases are attributed to male factors [33]. Risk factors for male infertility include varicocele, ageing, sexually transmitted diseases, lifestyle factors, and prolonged or intense exposure to certain types of toxins, chemicals, radiation, medications, and environmental toxicants [41,42]. All these factors can lead to a low count (oligospermia) or absence of sperm (azoospermia) and also to several morphological changes [42].
This medical condition is related to dysfunction of the pituitary gland and male gonads. Testicular functions include testosterone production and spermatogenesis, both being regulated by the hypothalamic-pituitary-gonad axis through a negative feedback system. This axis is further influenced by other endocrine organs, such as the adrenal gland and adipose tissue [43].
During spermatogenesis, gametes are created daily in the seminiferous tubules. All stages of the process are regulated by the endocrine system, specifically by T, E2, FSH, and luteinizing hormone (LH). The mechanisms that lead to male infertility are not completely clear, although changes in hormonal balance, particularly during sperm development, may affect sperm quality [44]. Its dysfunction, triggered by endocrine disruptors such as BPA, can result in the arrest or alteration of spermatogenesis, negatively affecting sperm production and quality [33,34]. Spermatogenesis is a complex process that involves genetic, hormonal, and environmental factors, and the failure of any of these can result in infertility. Recent studies have shown a decline in semen quality in men from several countries, including developing and developed countries, again emphasizing the role of environmental toxicants as a cause for this phenomenon [45].
In this section, we seek to find the possible connection of BPA exposure with several aspects of male infertility, incorporating data from epidemiological studies.

Evidence from Epidemiological Studies
In the testes, steroid hormones play an important role in spermatogenesis, semen production, and maintenance of secondary sexual characters and libido. They are also a target for endocrine disruptors [46]. Considering the endocrine disruption properties of BPA, an increase in its exposure in men may be associated with altered hormone levels and, therefore, with possible reproductive dysfunction [47]. In relation to the studies that characterise the parameters of semen quality, an analysis of the spermatogram or semen allows for evaluating the function of the testicle and has a prognostic value in the evaluation of fertility. The spermatogram is the cornerstone in the evaluation of male infertility [43].
In studies performed using animal models, BPA was suggested to be a testicular toxicant since it decreased sperm quality and motility, caused oxidative stress, and altered steroid synthesis [34][35][36][37][38]. Regarding the effects of BPA in humans, the data are still inconsistent, with limited evidence and no proven causality. Most of the studies observed an association between BPA exposure and sperm parameters, which may be related to male infertility. However, it is important to note that most of the studies are related to occupational and high levels of exposure [7,34].
The studies presented in this review are grouped according to evaluated parameters and main conclusions, including those with alterations in hormone levels, sperm parameters, hydroxymethylation, or no association. Note that in serum, the normal reference values are as follows: FSH of 1.5-12.4 mIU/mL, LH of 1.2-7.8 mIU/mL, TT of 2.5-8.4 pg/mL, prolactin of 2-18 ng/mL, and E2 of less than 40 pg/mL [48].
Since 2013, several studies have investigated the associations between BPA exposure in adulthood and fertility in different populations of men (e.g., occupational exposure, fertile or potentially infertile population, general population, and young people unaware of their fertility), but the results remain contradictory and inconclusive [49]. As in vivo human exposure is not feasible to assess the effects of BPA on male fertility due to ethical issues, the available evidence comes from observational epidemiological studies [50].
Three different studies evaluated occupational exposure to BPA in Chinese populations. The first, a cross-sectional study conducted by Zhou et al., evaluated the association between serum BPA levels and sex hormones. The sample included a total of 290 men, of whom 137 worked in a factory where they were exposed to BPA and 153 worked in a different factory not exposed to BPA. They found that increased serum BPA concentration was associated with a decreased free androgen index (FAI) and androstenedione (AD) and FT levels and increased sex hormone binding globulin (SHGB) levels [51]. In the second study, each of the 559 workers enrolled provided serum samples and were divided into two groups: exposed and unexposed to BPA. Both serum BPA concentrations and detection rates were higher in exposed men than in unexposed men, although the two groups showed no differences in serum levels of SHBG, total testosterone (TT), or inhibin B or AD. When comparing different durations of exposure (less and more than 5 years), a longer exposure was associated with increased serum BPA and SHBG levels and decreased AD levels. Briefly, chronic occupational exposure to BPA is associated with altered levels of male sex hormones and may affect male reproductive health [47]. Similar hormonal changes were also detected in a study by Liu et al. that recruited 592 workers, with and without occupational exposure to BPA, and collected urine and blood samples. The authors concluded that high exposure to BPA is associated with increased levels of prolactin, E2, and SHGB and a decrease in FSH, AD, and FAI [52]. In a similar perspective, a Japanese case-control study aimed to evaluate exposure to bisphenol A diglycidyl ether in epoxy resin workers. The results showed that bisphenol A diglycidyl ether can generate BPA endogenously with higher BPA levels in the urine of the epoxy resin workers when compared to controls. Furthermore, the exposed workers had decreased FSH levels, while there was no difference between LH and FT levels compared to the controls [53]. These studies presented similar results, with increased BPA exposure being related to hormonal level alterations including, specifically, increased SHBG and decreased AD, FAI, and FSH, which may contribute to male infertility.
Using a representative sample of the general Danish population, Lassen et al. evaluated urine, semen, and blood samples from 308 men. The authors concluded that BPA exposure is associated with higher concentrations of T, LH, E2, and FT, as well as a lower percentage of progressive sperm. However, no association was detected for other specific parameters of semen [32]. Similar results were found in an Italian population, where increased urinary BPA levels were positively related to increased TT levels, but no relation was found for E2 [54]. In a more recent study, Liang et al. collected urine and blood samples from 560 men, aged between 18 and 55, recruited from a low-industrialised mountainous area of China. The authors investigated whether exposure to environmental BPA at a lower level was related to serum concentrations of gonadotropic hormones. The results showed that exposure to BPA is associated with increased serum LH and FSH levels in male smokers along with decreased serum TT levels in men with BMI > 25 kg/m 2 . These findings suggest that the effects of environmental exposure to BPA on hormone levels may be modified by tobacco smoke and BMI [55]. These differences might be due to the different populations analysed as well as their specific characteristics, including age, BMI, and tobacco.
From a different perspective, Joensen et al. investigated the association between BPA exposure and testicular function in young Danish men with (n = 65) and without (n = 130) mutations in the filaggrin gene (FLG). Within the FLG mutation carrier group, higher urinary BPA concentrations were found to be associated with higher serum T and E2 levels and lower FSH levels. Furthermore, those with higher urinary BPA values had lower sperm motility. None of these associations were evident in the control group. The changes found in testicular function were not only due to the differences in BPA exposure levels since urinary levels did not differ between the two groups [56].
An investigation by Den Hond et al. aimed to verify whether greater exposure to endocrine disruptors was associated with an increase in subfertility in men, for which they used semen, urine, and blood samples from 120 men from four fertility clinics in Belgium. The authors divided the men into two groups according to total motile count (TMC) (cases: TMC < 20 million and controls: TMC > 20 million) and concluded that increased BPA levels were associated with a decrease in serum T and a higher risk of subfertility [57]. On the other hand, in a study on fertile men (n = 375), exposure to low environmental levels of BPA was suggested to be associated with a modest reduction in markers of free testosterone (FT), but any effects on reproductive function were considered to be small and of uncertain clinical significance [58].
More recently, a cross-sectional study also evaluated the association between urinary BPA concentration, semen quality, and reproductive hormone levels. The sample population included 215 healthy young university students (18-23 years old) from Spain [49], in which there was a reduction in the capacity of Leydig cells (increased LH levels) and a decrease in sperm count. If confirmed, these findings suggest that environmental BPA levels may negatively affect endocrine function in young people. However, no significant associations were found between BPA and other semen parameters or levels of reproductive hormones, contrary to the studies cited above [49]. Using the same cohort, during the Murcia Young Men's Study (Spain), 158 healthy participants (18-23 years old) were gathered to evaluate urinary BPA concentrations and the sperm DNA fragmentation (SDF) index. There was no association between urinary BPA concentrations and the SDF index in the total group. However, in the subgroup of men with an SDF index > 30%, positive associations were observed. The authors reported that, if confirmed, the results obtained suggested that environmental levels of BPA may have a negative impact on sperm function [59].
From the studies presented so far, despite some contradictory results, it is possible to assume that exposure to BPA may alter the concentration of several hormones, thus affecting male reproductive function, which can culminate in infertility.
Some investigations were conducted to analyse BPA concentrations and semen parameters in men already diagnosed with infertility. In a study involving 149 couples from an infertility clinic undergoing their first or second intracytoplasmic sperm injection procedure (ICSI), urinary BPA concentration in male partners and its relationship with semen quality parameters were evaluated. The authors found an association between increased BPA concentration and lower sperm count, sperm concentration, and sperm vitality [60]. After examining 190 men recruited from an infertility clinic, Meeker and co-workers also showed a decrease in sperm concentration and motility and morphology changes associated with urinary BPA concentration [61]. Similar results were also found in men (n = 218) exposed to BPA in the workplace, with decreased sperm concentration, total count, vitality, and motility; however, there was no association with morphology [62].
La Rocca et al. analysed BPA levels in the serum of 70 infertile and 83 fertile men from Italian metropolitan, urban, and rural areas. The purpose was to assess whether residential areas, with different expected levels of BPA, had different outcomes of potential infertility. The results showed that, when compared to the participants of urban and rural areas, infertile men from the metropolitan area had significantly higher levels of BPA, which were directly correlated with increased gene expression of ERα, ERβ, AR, aryl hydrocarbon receptor (AhR), and pregnane X receptor (PXR). These findings reinforce the concept that the living environment determines the main pattern of exposure to endocrine disruptors in the population and that the nuclear receptors panel in peripheral blood mononuclear cells is a potential biomarker that can be used to assess the impact of ED on reproductive health [63]. However, in a Chinese less industrialised area with lower exposure to environmental contaminants, BPA was related to decreased sperm quality. The aim of that cross-sectional study, which was conducted by Ji et al., was to examine the association of urinary BPA concentrations and sperm parameters related to movement. The participants included 500 fertile men from China who provided urine for BPA measurement and semen for analysis with a computer-aided sperm analysis (CASA) system. From the results obtained, the authors concluded that environmental exposure to BPA in a less industrialised area, where the levels of BPA in urine are lower, is related to decreased sperm concentration and motility parameters and increased velocity rates [64]. Scientific knowledge indicates that semen quality is a dominant component in determining male fertility. Current evidence from in vitro and human studies [65][66][67] show that a reduction in spermatozoa curvilinear velocity (VCL) and the amplitude of lateral head displacement (ALH) and an increase in linearity (LIN) are related to decreased male fertility. Thus, BPA-induced reductions in sperm concentration and ALH and increases in LIN can lead to a deterioration of male fertility. Changes in spermiogenesis and poor sperm movement may explain male subfertility resulting from BPA exposure, although the underlying mechanisms are still unknown [64].
In a different study, Omran and colleagues aimed to identify any kind of association between urinary BPA levels and infertility-related factors in Egyptian male patients. The study population consisted of 50 infertile men and 50 healthy men as controls. Semen quality parameters (concentration, count, morphology, and motility) and oxidative stress were determined along with sperm DNA damage. Urinary BPA concentrations were found to be similarly increased both in the cases and controls, with a negative association with semen quality and antioxidant levels and a positive association with DNA damage [68]. Also investigating an Egyptian population, a case-control study was performed to evaluate the association between BPA levels and male fertility. After collecting urine and semen samples from all the participants, BPA levels, semen parameters, and hormone profiles were analysed. In addition to BPA concentrations being significantly elevated in infertility cases compared to controls, the results also showed a correlation between those levels and sperm motility and some reproductive hormones (FSH, LH, TT, and E2) [48]. These results are in agreement with those from a study by Lassen et al., where higher levels of LH, TT, and E2 were also found in Danish men [32].
In a cross-sectional study on 984 Chinese men from an infertility clinic, there was an association between higher exposures to BPA and deterioration in semen quality, i.e., concentration and total sperm count and progressive and total motility below the normal reference [69]. Increased concentrations of BPA were also observed in infertile men recruited from a reproductive endocrinology unit in Greece. In that study, 55 men with different causes of infertility were analysed for BPA concentrations, and the results were compared to 25 fertile men from the general population. However, there was no difference in BPA concentrations between infertile men and control or between the causes of infertility. Therefore, these results suggest that although male infertility cannot be attributed to BPA exposure, high concentrations of BPA may contribute to this cause [70].
Vitku et al. determined the levels of BPA and oestrogens in the blood plasma and seminal plasma of 174 men with different degrees of infertility, according to their spermatogram. There was a change in E2 metabolism and sperm count, confirming the existence of an inverse association between the concentration of seminal BPA and sperm quality [31]. Later, the same research group also analysed the effects of BPA on spermatogenesis and steroid synthesis. Using the same biological matrices, the authors found that the presence of increased BPA levels in the seminal plasma can contribute negatively to sperm quality, decreasing their number, concentration, and morphology. In addition, the results indicated that BPA influences the synthesis of gonadal and adrenal steroids in several stages, reduces the rate of testicular steroidogenesis, and increases the rate of synthesis in the adrenal gland. Further disruption of steroidogenesis by BPA possibly occurs at the level of E2 metabolism through suppression of its catabolism or through stimulation of aromatase expression and/or activity [46].
In a different manner, during a study including 315 Polish men under the age of 45, Radwan and colleagues analysed the relationship between BPA exposure and semen quality, sperm DNA damage, and aneuploidy. The population sample was recruited from a reproductive health clinic and had normal sperm concentrations. The study provided evidence that BPA exposure is associated with poorer semen quality, considering the observed increase in immature sperm, decrease in sperm motility, and dissection of sperm chromosomes [39]. On the other hand, after evaluating the quality of semen in relation to the concentration of BPA, Pollard and colleagues concluded that higher exposure to this substance is associated with abnormal morphology of the sperm tail; however, no association was found for BPA exposure regarding sperm count. In the investigation, a sample of 161 men aged between 18 and 40 years and with unknown fertility was recruited [71].
With the aim of analysing exposure to BPA and sperm DNA methylation changes, Chinese men occupationally exposed (n = 77) and not exposed (n = 72) to BPA were recruited for sample collection (urine, blood, and semen). It was concluded that exposed men had lower sperm concentration when compared to the control group, and higher urinary BPA levels were associated with a lower degree of sperm DNA methylation [72]. Similarly, Tian and collaborators used semen samples from 158 Chinese factory workers, of whom 72 had exposure to BPA and 86 had no occupational exposure. The data obtained indicated that occupational exposure to BPA is associated with changes in the hydroxymethylation of human semen, which may be one of the possible mechanisms underlying the adverse effects induced by BPA on male reproductive function [73]. In a different study, BPA exposure was also analysed for its association with sperm acetylcholinesterase gene hydroxymethylation. In that comparative study, 74 and 83 male workers in factories with and without occupational exposure, respectively, were selected to enrol and provide urine and semen samples. Increased exposure to BPA was found to be related to increased hydroxymethylation levels, which led the authors to conclude that BPA may have adverse effects on male fertility [74].
A different approach was conducted by Palak et al., who evaluated the association between BPA levels, steroid hormone concentration, and circulating miRNA levels. The researchers found the following associations: increased levels of BPA and reduced E2 and AD concentrations in the seminal plasma of azoospermic men compared to healthy controls; a negative correlation between BPA levels and sperm concentration and normal semen morphology; a significant positive correlation between BPA levels and miR-let-7a and miR-let-7c levels; and a negative correlation with miR-518f levels. These results led the authors to conclude that BPA can negatively affect sperm quality and act directly on seminal plasma, affecting the testicular environment [75].
Although most of the studies described show an association between BPA exposure and changes in male hormones or semen parameters, there are others that did not find any association. Contrary to the studies cited above, Chen et al., who investigated whether exposure to phenols was related to idiopathic male infertility in the general population, concluded that there was no evidence of the association between BPA exposure and male infertility [76].
In the same line of results also appears the LIFE study that aimed to estimate the association between BPA and 35 parameters of semen quality among men of reproductive age from Michigan and Texas, USA. The 418 men recruited from the general population were to avoid the clinical and/or professional context, as observed in many studies already conducted and previously mentioned. This exploratory analysis found no evidence corroborating an adverse relationship between total urinary BPA concentration and semen quality [77].
Interestingly, a study by Hu and colleagues evaluated the interaction between BPA exposure and obesity in semen quality, a factor that increases BPA toxicity in spermatogenesis. The sample consisted of 357 subfertile men recruited from hospitals in the city of Nanjing, China. However, opposite to some studies previously mentioned, there was no relationship between BPA levels and sperm parameters, including sperm concentration, total count, motility, and semen volume [78]. In another investigation including 146 couples undergoing in vitro fertilisation, BPA concentrations did not significantly affect sperm concentration or motility parameters [79].
Based on the Fetal programming of Semen Quality (FEPOS) cohort, Benson and colleagues hypothesised that BPA and its substitutes (BPS and BPF) were related to poor semen quality. After analysing urine and semen samples from 556 young participants (18-20 years), the authors found no association between bisphenol urinary levels and semen characteristics [80]. Similarly, no statistically significant association was found between BPA concentration in the urine and parameters of seminal fluid in 105 men attending a fertility centre [81]. Taking into account that these results are contradictory to many previous investigations, the authors of these studies considered that the lack of association was possibly due to the decreased levels of BPA exposure.
Most recently, an investigation with the aim to understand the possible differences in exposure to phthalates and BPA between two male populations with different degrees of fertility concluded that there were no differences between urinary BPA levels in both groups. This population consisted of 155 men with changes in fertility and 211 controls, recruited in Milan, Italy [44].
From a different perspective, considering that pregnancy is one of the most vulnerable and sensitive periods, prenatal exposure to environmental chemicals has been widely studied and related to adverse health effects in the life of the mother and the offspring. EDCs can interfere with hormone homeostasis and signalling pathways that withstand pregnancy physiological changes, thus increasing maternal susceptibility and entailing risks not only for the mother but also for the newborn during childhood, adolescence, and adulthood [28]. Thus, two studies investigated maternal BPA exposure and reproductive male function. The Western Australian Pregnancy Cohort (Raine) Study is a large cohort designed to evaluate the effects of prenatal events on child and adult health. In this case, Hart and colleagues used this cohort to analyse how maternal BPA exposure affected male reproductive function later in life (20-22 years). The results showed a weak association between prenatal BPA exposure and testicular function, sperm concentration, and motility [82]. Similarly, in a Danish birth cohort study including 101 young men (18-20 years), there was also a positive association between high maternal exposure to BPA and reduced Leydig cell function; however, no association was found between anogenital distance and semen quality [83].
Overall, despite some contradictory results, most studies show that there is indeed a relationship between increased levels of BPA and semen quality, either directly or indirectly through changes in hormone levels. However, more studies are needed to understand the mechanisms involved in the contribution of BPA exposure to male infertility. Table 1 lists all the epidemiological studies mentioned above and their general conclusions.

Conclusions
With the industrialisation of modern society and the need for more convenient and practical solutions to keep up with the hectic lifestyle of today's families, plastic has become the preferred material for food and water containers. Human exposure to BPA is currently of great concern because this chemical has become so widespread in the environment and because it is detected in human biological fluids. Given the numerous sources and endocrine disruption potential of this chemical, it seems advisable to introduce worldwide biomonitoring in order to assess the risk to human health in detail. This monitoring can be a valuable tool in studies looking for relationships between BPA exposure and the prevalence of certain hormonal disorders.
The harmful effects on male fertility have been clearly illustrated in animal-based studies; however, human studies remain uncorroborated. In hormonal studies, the endpoints measured were typical: testosterone, oestradiol, follicle-stimulating hormone, luteinizing hormone, sex hormone-binding globulin, and/or androstenedione. In many of the studies analysing hormone levels, high exposure to BPA was associated with lower levels of testosterone compared with the normal reference values and with the respective controls. It was also related to changes in other hormone levels that may also contribute to male infertility. Human studies suggest that exposure to this substance is associated with changes in hormone levels and decreased sperm quality (motility, concentration, count, and vitality) and increased DNA damage and changes in sperm morphology, especially when there is high occupational exposure ( Figure 5). hygiene (e.g., not eating in the workplace, bathing after leaving work, etc.) to avoid as much as possible high exposure in the body. World governments should take measures (setting concentration limits, eliminating the use of certain substances, and tight control of agents already identified as causing reproductive harm) to ensure the precautionary principle. Furthermore, new and safer substitutes should also be created and tested, considering that those already replacing BPA have been found to have endocrinedisrupting properties as well, thus imposing a similar or even greater health risk as BPA.
Given the large-scale production of BPA, its constant presence in our daily lives, and the presumed effect on male fertility, measures should be taken to replace it with innocuous components and restrict its use.
In this review, only BPA is addressed; however, many other endocrine disruptors are ubiquitous in the environment and coexist in our body, which can have potentially synergistic effects on the quality of semen and reproductive hormones. The widespread use of bisphenols is undoubtedly dangerous for testicular function, and a reduction in their use is necessary to preserve male sexual and reproductive health.   However, the research available so far is insufficient to draw a definitive conclusion about BPA exposure and its effects on human male infertility. This lack of consensus and the contradictory results are because existing studies are limited, with mostly cross-sectional designs, small sample sizes, variety in groups of men (genetic predisposition, environmental conditions), and other methodological weaknesses. No type of epidemiological study is free of limitations, for example, cohort studies are expensive and require follow-ups, case-control studies are susceptible to information bias and have several external variables, and cross-sectional studies are subject to the time of data assembly, and causality cannot be assessed [84]. The majority of the studies discussed used a cross-sectional design, and there is a great lack of experimental approaches, thus, there is an urgent need to combine prospective cohorts, which encompass a large sample size, and experimental studies. Despite being detected in different biological samples, urine has the most advantages since it is easy to collect and non-invasive and has higher volume and BPA concentrations. However, BPA is rapidly metabolised and excreted in the urine, and although a single sample may reflect prolonged exposure, more samples per participant and throughout a period of time should be used as a strategy to obtain more reliable results. Furthermore, variety in the population samples is also a weakness. In addition to presenting different genetic predispositions and daily life habits, the studies group men from a large age range and not by age group, considering that hormone levels vary over age.
Thus, further studies that overcome these weaknesses are needed to establish the adverse effects of BPA more precisely and to ascertain whether the findings found using in vitro studies and with animal models can also be verified with the same significance in men. It is necessary and extremely important to carry out more homogeneous studies to obtain more consistent and reliable data. The current ambiguous conclusions could be resolved if BPA's exact mechanism of action were known, which remains to be determined. It would also be interesting to try to devise efficient therapeutic strategies to reverse the negative impacts of BPA on reproduction; for this, it is recommended to study all possible targets (proteins) and their impacts.
Still, the importance of reducing BPA levels in the environment should be emphasized to safeguard male fertility. Workers exposed to BPA should increase their personal protection (e.g., wearing a mask, gown, etc.) and pay attention to personal hygiene (e.g., not eating in the workplace, bathing after leaving work, etc.) to avoid as much as possible high exposure in the body. World governments should take measures (setting concentration limits, eliminating the use of certain substances, and tight control of agents already identified as causing reproductive harm) to ensure the precautionary principle. Furthermore, new and safer substitutes should also be created and tested, considering that those already replacing BPA have been found to have endocrine-disrupting properties as well, thus imposing a similar or even greater health risk as BPA.
Given the large-scale production of BPA, its constant presence in our daily lives, and the presumed effect on male fertility, measures should be taken to replace it with innocuous components and restrict its use.
In this review, only BPA is addressed; however, many other endocrine disruptors are ubiquitous in the environment and coexist in our body, which can have potentially synergistic effects on the quality of semen and reproductive hormones. The widespread use of bisphenols is undoubtedly dangerous for testicular function, and a reduction in their use is necessary to preserve male sexual and reproductive health.